Field performance of three mosquito collection methods for assessing the entomological efficacy of dual-active ingredient long-lasting insecticidal nets

Selection of mosquito collection methods is of crucial importance to evaluate the impact of vector control tools on entomological outcomes. During a cluster randomised control trial evaluating the relative efficacy of two dual-active ingredient (a.i.) long-lasting insecticidal nets (LLINs) compared to pyrethroid-only LLINs, we assessed the performance of different mosquito collection methods: Human landing catches (HLC), Centers for Disease Control and Prevention (CDC) light traps, and pyrethrum spray catches (PSC). Anopheles mosquitoes were collected using three collection methods in 4 houses, in each of the 60 trial clusters at baseline and every quarter for 24 months using PSCs and HLCs, while CDC light traps were performed during two quarters only. Mean density of vectors collected per method per night was the highest with HLCs (15.9), followed by CDC light traps (6.8); with PSCs (1.1) collecting 10 times less mosquitoes than HLCs. All three collection methods collected fewer mosquitoes in the Interceptor G2® dual a.i. arm, compared to the other trial arms, although only HLCs and PSCs demonstrated strong evidence of this due to a greater number of collection rounds undertaken, than CDC light traps. The broadly similar results regarding the differential impact of the two dual a.i. LLINs showed by the three collection methods suggest that the more ethically acceptable, cheaper, and logistically simpler methods such as CDC light traps could be prioritised for use in large community trials for measuring the efficacy of vector control tools.

Mosquito sampling. One data collection round occurred at baseline in September and October 2019 before the net distribution, while 8 post-net distribution rounds were conducted between June 2020 and April 2022. HLC and PSC collections were conducted in all 60 clusters every 3 months. CDC light traps were only conducted at baseline and during two round post-net distribution (16-and 19-months post-net distribution (June-July, and September-October 2021, referred to as Rounds 5 and 6)). For HLC sampling, one house was selected randomly in each cluster, with three further houses selected within 20 m of the randomly selected house (for a total of 4 houses per cluster per round). For PSC sampling, four houses close to the HLC selected houses were used. At baseline, one CDC light trap was placed in a house neighbouring a house used for HLC. The other three houses for CDC light traps were picked randomly in the cluster. Post-net distribution, the CDC light traps were used in houses neighbouring the four houses used for HLCs, similar to the PSC sampling method. There was a total of 240 collection points per round for each collection method.
HLCs were conducted with one trained adult volunteers positioned inside each house. The first group of volunteers collected mosquitoes from 19:00 pm to 01:00 am and were replaced by a second group of collectors from 01:00 to 07:00. Collections were done using hemolysis tubes and a flashlight to collect mosquitoes that landed on their lower limbs (Fig. 2a).
Standard miniature CDC light traps (Model 512; John W. Hock Company, Gainesville, FL.) were set from 19:00 pm to 7:00 am at the foot of an occupied bed fitted with a mosquito net (a study net or other net) positioned www.nature.com/scientificreports/ around 70 cm above the floor (Fig. 2b) 9 . In the morning, trapped mosquitoes were transferred to a paper cup and taken back to the laboratory. In houses selected for PSCs, white sheets were installed to cover the floor and furniture and an insecticide aerosol combining 0.25% transfluthrin and 0.20% permethrin (trade name Rambo) was sprayed (Fig. 2c) for 15 s Figure 1. Map of the study area showing the three study arms, each with 20 clusters. The map was produced by study investigators (M.A., E.D., J.C.) with Esri ArcGIS Pro 3.1 software (https:// pro. arcgis. com/ fr/ pro-app/ latest/ get-start ed/ downl oad-arcgis-pro. htm) using study data and online data provided by GADM (https:// gadm. org/ downl oad_ count ry. html) and Natural Earth (https:// www. natur alear thdata. com/). www.nature.com/scientificreports/ per room. Door and windows were shut and after fifteen minutes, all mosquitoes that had fallen on the sheets were recovered with forceps and stored in petri dishes. All collections were performed in households located in the core area of each cluster to prevent contamination from adjacent clusters that might have received different nets.
Mosquito processing. Mosquitoes from the three collection methods were identified to species level using the morphological identification key of Gillies and Meillon 33 .
A subset (approximately 30%) of An. gambiae s.l. collected with HLCs and CDC light traps were used to assess P. falciparum sporozoite infection by ELISA CSP on head and thoraces 34 . Sporozoite positive mosquitoes and a subset of negative mosquitoes caught by HLCs and CDC light traps were then used for molecular species identification, using the protocol described by Santolamazza et al. 35 Data analysis. The nightly indoor vector density was calculated at the household level for each of the mosquito collection methods (HLCs, PSCs, and CDC light traps), by dividing the total number of An. gambiae s.l. collected by the total number of collector night. The SR was determined by dividing the number of sporozoite  www.nature.com/scientificreports/ positive mosquitoes by the total tested. The nightly entomological inoculation rate (EIR) was calculated by multiplying the mean nightly indoor vector density by the SR at the cluster level. For comparison of mosquito density between collection methods, mixed effect generalised linear model with a negative binomial distribution was used for mosquito density, adjusting for study district (Cove, Ouinhi and Zaganando) as a fixed effect and including study arm as a fixed effect in the post-net distribution data. Cluster was included as a random effect in all models, with collection round also included as a random effect in post-net distribution models. A similar strategy was used with a logistic regression model for SR. Vector density and SR were compared between collection methods using HLCs as the reference.
For comparison of mosquito density and EIR between study arms, mixed effect generalized linear models with a negative binomial distribution, with collection round and cluster included as random effects were used. A mixed effect logistic regression model, with collection round and cluster included as random effects, was used to compare SR. Each collection method was assessed separately.
All data analyses were performed using the Stata 15.0 (Stata Corp., College Station, TX) software.
Ethical considerations. Ethical approvals were issued by Benin's National Ethics Committee for Health Research (N°30/MS/DC/SGM/DRFMT/CNERS/SA, Approval n°6 of 04/03/2019), and the Ethics Committee of the London School of Hygiene and Tropical Medicine (16237-1), after reviewing the study protocol. All study participants gave their informed consent, prior to their involvement. Only trained collectors who were able to capture mosquitoes before they got bitten, were involved in the study. Before the beginning of the study, they were vaccinated against yellow fever, and treated when found positive for malaria infection. During the trial, care was provided to them at the local health facility whenever they had symptoms similar to those of malaria. All methods were performed in accordance with the relevant guidelines and regulations.

Results
Performance comparison of collection methods. Number Fig. 3). At baseline, a higher proportion of An. coluzzii were caught with HLCs (50.9%) than with CDC light traps (11.7%). Post-net distribution, An. coluzzii formed two thirds of the total tested mosquitoes for both HLCs and CDC light traps (66% for both collection methods with pooled data: cluster range for HLCs (0.7-98.1%); cluster range for CDC light traps (0-100%). The rest of the Anopheles species tested were An. gambiae s.s. for both baseline and post-net distribution periods ( Fig. 4).
At baseline, in all three collection methods, at least one third of mosquitoes caught were Culex spp. (between 32.1 and 38.0%). The proportion of Culex caught was reduced post-net distribution in all three collection methods, to around a quarter of the collection [23.8% for HLCs, 25.4% for PSCs, and 22.1% for CDC light traps* (*in two rounds only)] (Fig. 3).
At baseline, Mansonia spp accounted for 34.7%, 40.2%, and 28.8% of each collection for HLCs, CDC light traps and PSCs, respectively. Post-net distribution, the proportion of Mansonia increased for HLCs (45.1%) and CDC light traps (54.4%) (in two rounds only) but remained similar to baseline for PSCs (26.9%). Overall, Mansonia mosquitoes were more likely to be caught by HLCs and CDC light traps compared to PSCs (Fig. 3).
Overall, Aedes spp. were sampled by the three collection methods at low frequencies (< 4%). Aedes spp. was mostly collected through HLCs and PSCs. Other mosquitoes caught in very lower proportions (< 0.07%) included Coquillettidia and Eretmapodites.
Density and SR of An. gambiae s.l collected at baseline and post-net distribution through HLCs, CDC light traps, and PSCs. A total of 10,093 specimens of An. gambiae s.l. were collected at baseline and 11,465 post-net distribution with all collection methods. At baseline, the mean indoor density of An. gambiae s.l. was significantly higher with HLCs [ (Table 1). However, the reduction in density observed in mosquitoes caught using PSCs between each of the intervention arms compared to the control arm was of similar magnitude as the HLCs (Table 2) Overall, the reduction in An. gambiae s.l. was significant using PSCs in the pyriproxyfen-pyrethroid LLIN arm. This effect was particularly clear in the first year of the study. Overall, there was no strong evidence for a reduction in mosquito density in the pyriproxyfen-pyrethroid LLIN arm using HLCs. Similar conclusions regarding the efficacy of the chlorfenapyr-pyrethroid LLIN arm could be made for both HLCs and PSCs, with a much more pronounced reduction in vector density in the chlorfenapyr-pyrethroid LLIN arm, compared to the pyrethroid-only LLIN arm, irrespective of the mosquito collection method used. This was observed during the first and second year of the trial, with both HLCs and PSCs (Table 2). Table 1. Density and SR of An. gambiae s.l collected at baseline and post-net distribution (rounds 5 and 6) through HLCs, CDC light traps* (*density only), and PSCs. N of An, number of An. gambiae s.l.; IRR, incidence rate ratio; CI, confidence interval; SR, sporozoite rate; aOR, adjusted odd ratio; n/a, no data; b/p/n, bite/person/night. Density and SR were adjusted for study area for baseline and post-net distribution. Study arm included as a fixed effect in the post-net distribution model. Cluster was included as a random effect in all models. Collection round was included as a second random effect in the post-net distribution model. 2.5 0.07 0.06-0.08 < 0.001 n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a

Discussion
The comparison of three mosquito collection methods (HLCs, CDC light traps and PSCs) as part of a large RCT testing dual-a.i. LLINs in Benin showed that HLCs resulted in far greater numbers of mosquitoes being collected as compared to CDC light traps and PSCs. Overall, the mean density of An. gambiae s.l. collected was half with CDC light traps compared to HLCs, while it was 10 times lower with PSCs. However, despite this difference, CDC light traps and PSCs often measured similar reductions in mosquito density, SR and EIR* (*CDC light traps only) as compared to HLCs between study arms.
The higher Anopheles densities caught with HLCs compared to CDC light traps in the present study has been also reported in several other studies 10,17 . In those studies HLCs caught 1.5 to 1.7 times more Anopheles vectors than CDC light traps; in the present study HLCs caught 2.2 (95% CI 2.1-2.3) times more (Table 1). This shows there was no consistent difference in density between HLCs and CDC light traps, as previously found by Briët et al. 36 after reviewing 13 studies. One of the possible reasons for the higher vector densities with HLCs compared to CDC light traps, could be due to the fact that numerous stimuli (olfactory, visual cues, volatiles, body heat, and humidity) involved in the collections using human bait exert a strong attraction on host-seeking mosquitoes, while only visual ones are used during CDC light trap collections. Moreover, the study LLINs which sleepers were under and which were installed close to the CDC light traps might have repelled some of the host seeking mosquitoes, so reducing their density as previously observed by Kirby et al. 37 .
In the present study, PSCs resulted in a far lower (at least 10 (95% CI 9.8-11.7) times) mean indoor vector density compared to HLCs (Table 1), as previously observed by Ndiath et al. 10 Originally, this method was used to sample mosquitoes that rest inside houses and therefore cannot be considered a quantitative method for evaluating indoor vector density, as mosquitoes might have left the houses early in the morning to seek for outdoor hosts due to unsuccessful blood meal intake that occurred overnight. The latter observation might be  www.nature.com/scientificreports/ due to a possible repellent effect induced by the insecticides incorporated in the trial LLINs that also constituted a physical barrier that reduced the host-vector contact as previously observed by Sovi et al. 38 In addition, in the Cove, Ouinhi and Zagnanando districts where malaria vectors were highly pyrethroid-resistant with several resistance mechanisms 31 , it is possible that the aerosol insecticide used for the PSCs failed to knock down or kill all resting mosquitoes, resulting in a lower density of collected samples. Finally, the lack of standardization (different collection times from cluster to cluster, possible dispersion of mosquitoes in the presence of eaves or holes in the walls) for this method could also have had an impact on mosquito density caught using PSCs.
Conflicting results regarding SR have been found in previous studies with some finding SR was similar for both HLCs and CDC light traps 10,17 whilst others, found it was higher in CDC light traps compared to HLCs 16,39 . However, none of these trends was observed in the present study as the SR was 1.5 (95% CI 1.4-1.6) times higher with HLCs than with CDC light traps at both baseline and post-net distribution (Table 1). This might be due to the relative time in the physiological age structure of the vector populations, as collections occurred only once at baseline, and twice post-net distribution.
Both CDC light traps and HLCs were used to assess the effect of chlorfenapyr-pyrethroid and pyriproxyfenpyrethroid LLINs on key entomological indicators (density, SR, and EIR) of malaria transmission as compared to pyrethroid-only net. Overall, the two collection methods showed that the chlorfenapyr-pyrethroid LLIN tended to be more effective than the pyriproxyfen-pyrethroid LLIN, but with weak evidence, as there were only two rounds of collection (Table 3). This finding is reminiscent of the large RCT conducted by Mosha et al. 40 in Tanzania, using CDC light traps, that showed superior efficacy of chlorphenapyr-pyrethroid LLINs on vector density and EIR, which was not observed for pyriproxyfen-pyrethroid LLINs.
PSCs and HLCs showed a significant reduction in vector density in the chlorfenapyr-pyrethroid LLIN arm. By comparison, PSCs displayed a significant reduction in vector density in the pyriproxyfen-pyrethroid LLINs arm, while HLCs did not ( Table 2). This might be because PSCs caught a higher relative frequency of An. gambiae s.l. as compared to HLCs (44.6% vs 27.2%). This made it easier to see differential impact of the two dual-a.i. LLINs with PSCs, as compared with HLCs.
Overall, the same morphologically identified mosquito genera were collected using the three collection methods. The proportion of Culex mosquitoes decreased post-net distribution, compared to baseline in all collection methods. Post-net distribution, the proportion of Mansonia spp. increased in HLCs and CDC light traps but remained the same in PSCs as compared to baseline. For Anopheles mosquitoes, their proportion remained similar in HLCs and CDC light traps and increased in PSCs post-net distribution, as compared to baseline (Fig. 3). For the three mosquito genera, the changes in species composition observed post-net distribution in certain collection methods compared to baseline could be due to the seasonality. Indeed, the baseline collection occurred at only one time point (short rainy season: September-October 2019), so could not capture a representative image of the species composition compared to the post-net distribution period which occurred over 8 time points (all seasons: June 2020 to April 2022). From baseline to the post-net distribution period, changes in Mansonia and Anopheles species composition followed the same trend in HLCs and CDC light traps, while it did not in PSCs. This might be because both HLCs and CDC light traps collect host seeking mosquitoes 41 , while PSCs sample indoor resting ones 10 .
At the molecular species level, the composition was similar post-intervention for HLCs and CDC light traps as observed by Mawejje et al. 17 However, relative molecular species composition was different between HLCs and CDC light traps at baseline, with An. gambiae s.s. mostly collected through CDC light traps, and in similar proportions to An. coluzzii through HLCs (Fig. 4). This might be due to the difference in how sampled houses for both collection methods have been selected between baseline and post-net distribution period. The decline in the proportion of An. gambiae s.s. observed post-net distribution compared to baseline for both HLCs and CDC light traps (Fig. 4), suggests that the intervention tools implemented were more effective on this mosquito species, which was not the case for An. coluzzii.
Limitations for the present study include the only two post-net distribution rounds of data collection performed with CDC light traps, the lack of molecular (species identification, and P. falciparum sporozoite infection) data for mosquito collected through PSCs, the non-inclusion of environmental conditions (temperature, humidity) as fixed effects in the analyses to account for overtime variations, as well as the fact that the trial was conducted in a single region, which limits the generalization of the findings.
To our knowledge, the present study is the first one to evaluate whether PSCs and CDC light traps are equally as effective as HLCs in estimating the differential impact of dual-a.i. LLINs on key entomological indicators of malaria transmission. Overall, HLCs collected more mosquito vectors than CDC light traps and PSCs, so remain the gold standard method to be used for entomological monitoring, notwithstanding the ethical issues involved and the fact that in the daily life, nobody in the community is going to sit and expose their lower limbs to receive potential mosquito bites overnight-so the mosquitoes caught might not be reflective of a realistic risk of biting. However, CDC light traps and PSCs were found to be as effective as HLCs in evaluating the differential impact of dual-a.i. LLINs on key entomological indicators of malaria transmission as compared to pyrethroid-only nets.

Conclusion
In our study area, HLCs may be prioritized for single cross-sectional studies, as this collection method tends to collect more mosquitoes. However, due to the ethical issues raised by this collection method, CDC light trap which is a considerably simpler, cheaper and logistically easier method that also collected host-seeking mosquitoes, could be used as an alternative option, as part of longitudinal entomological studies given it showed potential in assessing the differential impact of next-generation vector control tools.
PSC that collected a much lower number of indoor resting vectors, was also able to evaluate the efficacy of next-generation nets compared to standard pyrethroid-only nets. www.nature.com/scientificreports/

Data availability
The datasets generated and/or analysed during the present study are available on reasonable request from the corresponding authors.